In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24

摘要

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19* 23 (V-max 111.5 +/- 16.0 pmol/min/mg, K-m 158.3 +/- 88.0 mu M) protein relative to CYP2 C19 WT (V-max 101.6 + 12.4 pmol/min/mg, K-m 123.0 +/- 19.2 mu M) protein had no significant difference. In contrast, the K-m of CYP2C19* 24 (270.1 +/- 57.2 mu M) increased significantly as compared to CYP2C19 WT (123.0 +/- 19.2 mu M) and V-max of CYP2C19* 24 (23.6 +/- 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 +/- 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint = V-max/K-m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19